Abstract ESCRT protein complexes play essential roles in membrane remodeling for multivesicular body formation, intercellular communication, formation of the nuclear envelope, and cell division. We recently showed that the obligate intracellular parasite Toxoplasma gondii repurposes host ESCRT machinery to acquire macromolecular resources from infected cells. Host-derived resources critically fuel rapid growth of T. gondii acute stage parasites, which are responsible for debilitating disease in immunodeficient or congenitally infected individuals. Although we identified one parasite effector protein (GRA14) that directly binds the ESCRT-I complex protein TSG101 and recruits it to the parasite’s replicative compartment, this protein only accounts for ~50% of TSG101 recruitment. Using a novel combination of CRISPR/Cas9 screening, high content imaging, and machine learning- driven image analysis, in addition to GRA14, we identified 7 new parasite secretory proteins that putatively contribute to TSG101 recruitment. Among the newly identified parasite proteins was ROP18, which is a major virulence factor that is best known for effector function in evading innate immunity. Importantly, ROP18 deficient parasites showed significantly less recruitment of TSG101, thus validating it as a hit and exposing a novel function for this parasite kinase. However, additional studies are necessary to determine how ROP18 contributes to TSG101 recruitment and which of the other 6 hits function in such recruitment. To meet this need, in Aim 1 we will determine if the kinase activity of ROP18 is required for TSG101 recruitment to the PV and identify ROP18-linked parasite and host proteins that regulate TSG101 recruitment. In Aim 2, we will validate other hits from the screen and determine if they are working in conjunction with GRA14 or ROP18. Together, these studies will identify a novel protein network responsible for engaging host ESCRT machinery to ensure the successful replication of an important human pathogen.